The present invention relates to transgenic plants with increased resistance to geminivirus infection, and the mutants of the AL1/C1 (Rep) geminivirus protein useful for producing such plants. Methods of screening for suitable mutants are also provided.
The geminiviruses are a large and diverse family of plant DNA viruses, with circular single-stranded (ss) DNA genomes that replicate through circular double stranded DNA intermediates. See Hanley-Bowdoin et al., Cri. Rev. Plant Sci. 18:71 (1999); Lazarowitz, Crit. Rev. Plant Sci. 11:327 (1992); Timmermans et al., Annu. Rev. Plant Physiol. 45:79 (1994). Viral DNA replication, which results in both single and double stranded viral DNAs in large amounts, involves the expression of only a small number of viral proteins that are involved in either replication or viral transcription. The geminiviruses appear to rely primarily on the machinery of the host to copy their genomes and express their genes, including the nuclear DNA and RNA polymerases of their plant hosts. These properties of geminiviruses are unusual among plant viruses, most of which are RNA viruses or replicate through RNA intermediates using virus-encoded replicases. Geminiviruses infect a broad variety of plants and cause significant crop losses worldwide.
Geminiviruses are subdivided on the basis of host range in either monocots or dicots, genome structure, and insect vector. Subgroup I geminiviruses (also known as Mastreviruses) are transmitted by leafhoppers and infect primarily monocots, although Subgroup I geminiviruses that infect dicots are known. Subgroup II geminiviruses (also known as Curtoviruses) are transmitted by leafhoppers and infect dicots. Subgroup III geminiviruses (also known as Begomoviruses) are transmitted by whiteflys and infect dicots. Subgroups I and II viruses have genomes comprising a single ssDNA component; Subgroup III geminiviruses typically have a bipartite genome comprising two similarly sized DNAs (usually termed A and B), as illustrated by African cassava mosaic virus (ACMV), tomato golden mosaic virus (TGMV) and potato yellow mosaic virus. However, monopartite geminiviruses that infect dicots are known, for example Tomato Yellow Leaf Curl Virus (TYLCV). The genomes of monopartite Subgroup II and III geminiviruses have an arrangement of genes similar to the AL1, AL2 and AL3 genes found on the A DNA component of bipartite Subgroup III geminiviruses.
Subgroup II viruses are also divided into xe2x80x9cold worldxe2x80x9d and xe2x80x9cnew worldxe2x80x9d viruses, a division based on evolutionary divergence.
For successful infection of plants by bipartite geminiviruses, both the A and B genomic components are required. Sequence analysis of the two genome components reveals six open reading frames (ORFs); four of the ORFs are encoded by DNA A and two by DNA B. On both components, the ORFs diverge from a conserved 230 nucleotide intergenic region (common region) and are transcribed bidirectionally from double stranded replicative form DNA. The ORFs are named according to genome component and orientation relative to the common region (i.e., left versus right (L/R), or virion versus complementary sense (V/C)). Certain proteins are known to be involved in the replication of viral DNA (REP genes). See, e.g., Elmer et al., Nucleic Acids Res. 16:7043 (1988); Hatta and Francki, Virology 92:428 (1979).
The A genome component contains all viral information necessary for the replication and encapsidation of viral DNA, while the B component encodes functions required for movement of the virus through the infected plant. The DNA A component of these viruses is capable of autonomous replication in plant cells in the absence of DNA B when inserted as a greater than full length copy into the genome of plant cells, or when a copy is transiently introduced into plant cells. In monopartite geminivirus genomes, the single genomic component contains all viral information necessary for replication, encapsidation, and movement of the virus.
Geminiviruses cause substantial losses among economically important crops, including tomato, bean and cucurbit. Current strategies to control geminivirus infections target the insect vectors that carry the viruses. However, the use of insecticides to control or combat a geminivirus infection can be expensive and inefficient. Additionally, insect hosts may vary in their susceptibility to available insecticides, and resistance to insecticides may develop over time. See Markham et al., Pestic. Sci. 42:123 (1994).
Varied approaches have been used in attempts to generate geminivirus-resistant plants, including classical breeding and transgenic approaches, with limited success. Unlike plant RNA viruses, the introduction of geminivirus sequences into transgenic plants does not confer resistance and, conversely, frequently results in the production of functional viral proteins (Hayes and Buck, Nucleic Acids Res. 17:10213 (1989); Hanley-Bowdoin et al., Proc. Natl. Acad. Sci. USA 87:1446 (1990)). Kunik et al. report transgenic tomatoes that contain a geminivirus coat protein gene (Kunik et al., BioTechnology 12:500 (1994)). Expression of antisense RNAs against geminivirus replication proteins in transgenic plants reduces the level of viral DNA accumulation up to 70% (Day et al., Proc. Natl. Acad. Sci. USA 88:6721 (1991)), to a level that is still sufficient to confer wild type viral symptoms (Hanley-Bowdoin et al., Plant Cell 1:1057 (1989)). Similarly, the presence of defective-interfering replicons in transformed plants can reduce the level of viral DNA accumulation by about 70% (Frischmuth and Stanley, Virology 200:826 (1994)). The antisense RNAs and defective-interfering replicons function best against their cognate viruses (Bejarano et al., Plant Mol. Biol. 24:241 (1994)), further limiting their usefulness. Antisense RNA targeted to mRNA of the Rep protein (encoded by the C1 gene) was used by Bendahmane and Gronenborn to produce transgenic Nicotiana benthamiana plants with altered responses to TYLCV. Plant Mol. Biol. 33:351(1997)
Accordingly, it is desirable to devise new strategies to control geminivirus infection.
A first aspect of the present invention is a plant comprising transformed plant cells, said transformed plant cells containing a heterologous nucleic acid construct comprising, in the 5xe2x80x2 to 3xe2x80x2 direction, a promoter operable in said plant cells, a nucleic acid sequence encoding a mutant AL1 protein, where said nucleic acid sequence is located downstream from said promoter and operatively associated therewith, and comprising a mutation in the Rb binding region, whereby binding of said mutant AL1 protein to a plant Rb protein is reduced compared to binding which would occur in the presence of a wild-type AL1 protein; and a mutation in the AL1 protein, whereby said mutant AL1 protein suppresses viral replication compared to that which would occur in the presence of a wild-type AL1 protein; and a termination sequence positioned downstream from said nucleic acid sequence and operatively associated therewith, wherein expression of said mutant AL1 protein increases resistance of said plant to infection by at least one geminivirus, compared to a non-transformed control.
A further aspect of the present invention is a method of making a transgenic plant having increased resistance to geminivirus infection. The method comprises providing a plant cell capable of regeneration; transforming the plant cell with a DNA construct comprising, in the 5xe2x80x2 to 3xe2x80x2 direction, (a) a promoter operable in said plant cells, (b) a nucleic acid sequence encoding a mutant AL1 protein, said nucleic acid sequence located downstream from said promoter and operatively associated therewith, and comprising i) a mutation in the Rb binding region, whereby binding of said mutant AL1 protein to a plant Rb protein is reduced compared to binding which would occur in the presence of a wild-type AL1 protein; and ii) a mutation in the AL1 protein, whereby said mutant AL1 protein suppresses viral replication compared to that which would occur in the presence of a wild-type AL1 protein; and (c) a termination sequence positioned downstream from said nucleic acid sequence and operatively associated therewith; and then regenerating a transgenic geminivirus-resistant plant from said transformed plant cell, wherein expression of said mutant AL1 protein increases resistance of said plant to infection by at least one geminivirus, compared to a non-transformed control.
A further aspect of the present invention is a nucleic acid construct comprising an expression cassette, which construct comprises, in the 5xe2x80x2 to 3xe2x80x2 direction, a promoter operable in a plant cell, a nucleic acid sequence encoding a mutant AL1 protein, said nucleic acid sequence located downstream from said promoter and operatively associated therewith, and comprising a mutation in the Rb binding region, whereby binding of said mutant AL1 protein to a plant Rb protein is reduced compared to binding which would occur in the presence of a wild-type AL1 protein; and ii) a mutation in the AL1 protein, whereby said mutant AL1 protein suppresses viral replication compared to that which would occur in the presence of a wild-type AL1 protein; and a termination sequence positioned downstream from said nucleic acid sequence and operatively associated therewith.
A further aspect of the present invention is a method of producing nucleic acid constructs useful in conferring increased geminivirus-resistance to plants, comprising, screening mutants of a geminivirus AL1 protein to identify mutations that suppress the ability of the AL1 protein to bind to a plant Rb protein; preparing a nucleic acid molecule encoding an AL1 protein having said mutation, and further having a mutation that suppresses geminivirus replication compared to that which would occur in the presence of a wild-type AL1 protein; and preparing a nucleic acid construct comprising, in the 5xe2x80x2 to 3xe2x80x2 direction, a promoter operable in a plant cell, a nucleic acid sequence encoding said mutant AL1 protein, said nucleic acid sequence located downstream from said promoter and operatively associated therewith, and a termination sequence positioned downstream from said nucleic acid sequence and operatively associated therewith.
A further aspect of the present invention is a nucleic acid construct comprising an expression cassette, which construct comprises, in the 5xe2x80x2 to 3xe2x80x2 direction: a promoter operable in a plant cell, a nucleic acid sequence encoding a mutant AL1 protein, said nucleic acid sequence located downstream from said promoter and operatively associated therewith, and comprising: a mutation in the oligomerization domain, whereby binding of said mutant AL1 protein to wildtype AL1 protein is reduced compared to binding which would occur with a wild-type AL1 protein; and a mutation in the AL1 protein, whereby said mutant AL1 protein suppresses viral replication compared to that which would occur in the presence of a wild-type AL1 protein; and a termination sequence positioned downstream from said nucleic acid sequence and operatively associated therewith.